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Significance of glutathione in medicine and pharmacy
Alterations in GSH/GSSG homeostasis and in GSH-dependent enzymes or processes have been widely reported to be correlated with several human diseases. Most of these pathological conditions are associated with depletion of GSH content which can lead to the progression of cellular death and cytotoxicity. GSH depletion has been shown to either predispose cells to apoptosis or directly trigger cell death in response to different apoptotic stimuli such as ROS generation (Franco, et al., 2007). However, protective effect of GSH in preventing cell death in the absence of any ROS formation is also observed (Deas, et al., 1997). Hence, the precise mechanism of apoptosis modulation by GSH still remains elusive. Nevertheless, shifting the GSH/GSSG redox toward the oxidizing state activates several signaling pathways, thereby progressing the pathogenesis of many diseases including cancer, neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease), HIV, AIDS, inflammation, kwashiorkor, liver disease, cystic fibrosis, infection, heart attack, stroke, ischemia, and metabolic diseases (diabetes, obesity) (Wu, et al., 2004, Franco, et al., 2007). On the other hand, in carcinogenous cells, high intracellular GSH concentrations associated with increased expression of GST and GSH-transporters are responsible for resistance to anticancer drugs (Franco, et al., 2007).
By its extraordinary antioxidant properties, GSH belongs to the group of molecules looking for clinical applicability. Since GSH deficiency is due to an aberration in efflux rather than synthesis, supplementation with GSH is desirable rather than the use of GSH precursors (Townsend, et al., 2003). As is the case of peptide and protein drugs, the clinical use of GSH is limited only to parenteral administration for approved indications such as the treatment of alcohol and drug poisoning and protection against toxicity induced by cytotoxic chemotherapy, radiation trauma, or AIDS-associated cachexia. Nevertheless, rapid enzymatic degradation brings the GSH plasma concentration to negligible levels within a few minutes after intravenous (iv) administration of high GSH doses (Trapani, et al., 2007).
Therefore, the development of dosage forms enabling GSH administration by alternative routes (such as oral or pulmonary) may provide increased clinical value to GSH. Due to GSH stability and uptake, inhalation methods appear quite promising. GSH in the epithelial lining fluid (ELF) of the lower respiratory tract is thought to be the first line of defense against oxidative stress involved in many respiratory tract diseases. Inhalation is the only known method that increases GSH levels in the ELF and exhibits the effectiveness for the treatment of several pulmonary diseases (Prousky, 2008).
To assess the feasibility of supplementing oral GSH, Witschi et al. (1992) have determined the systemic availability of GSH in 7 healthy volunteers. However, after a single GSH dose of 3 g, the concentrations of GSH, cysteine, and glutamate in plasma did not increase significantly, suggesting that the systemic availability of GSH is negligible in man.
Because of hydrolysis of GSH by intestinal enzymes, it is not possible to increase circulating GSH to a clinically beneficial extent by the oral administration of GSH (Witschi, et al., 1992).
In order to be administered by the oral route, GSH needs to be protected against both chemical (risk of oxidation of GSH to GSSG) and enzymatic hydrolysis during gastrointestinal transit. There have been only a few studies with oral GSH. In the work of Gate et al. (2001), GSH was incorporated into liposomes and GSH-loaded liposomes restored intracellular GSH in cells previously treated with buthionine sulphoximine (BSO), which is a γ-glutamylcysteine synthase inhibitor (Gate, et al., 2001). On the other hand, Trapani et al. (2007) have encapsulated GSH alone or in combination with hydroxypropyl-β-cyclodextrin in Eudragit RS 100 – based microparticles by an emulsification solvent evaporation method and evaluated this delivery system for an oral administration. In their report, it has been suggested that the system could be useful for the oral administration of GSH based on the in vitro enzymatic stability and frog intestinal permeability studies (Trapani, et al., 2007). Nevertheless, this GSH delivery system was not tested in animals.
From a pharmaceutical technology standpoint, it is worthy of note that GSH is able to act itself as a permeation enhancer by inhibition of protein tyrosine phosphatase activity via a disulfide bond formation. In this way, the tight junctions of the intestinal epithelium become open and paracellular permeability increases for hydrophilic macromolecular drugs (Bernkop-Schnurch, et al., 2003). Since GSH can be rapidly oxidized to GSSG on the surface of epithelial cells, better results have been obtained with the use of thiomer/GSH system. In this system, a thiolated polymer (thiomer) cooperates by reducing GSSG and remaining for a prolonged period of time at the site of absorption because of its mucoadhesive properties. The potential usefulness of this system is already supported by in vitro and in vivo studies e.g. for calcitonin, insulin or heparin (Clausen, et al., 2002, Bernkop-Schnurch, et al., 2003).
Analytical methods for glutathione detection and determination
High constant interest in the determination of the glutathione status (concentration of GSH, GSSG and other oxidized forms, and the ratio GSH/GSSG) in various cells, tissues and organs is related to the role of GSH in respect with its biochemical, physiological, toxicological and clinical aspects. As mentioned above, glutathione status constitutes a dynamic equilibrium between GSH synthesis, catabolism, transport, oxidation and reduction depending on cellular state and environmental conditions. Changes in glutathione status occur under both normal physiological situations and stresses, or result from genetic defects (e.g. E. coli mutants in genes encoding enzymes of GSH metabolism) or the action of some chemicals (e.g. BSO) (Smirnova & Oktyabrsky, 2005).
Generally, GSH is found in cells in high concentrations up to 10 mM in most cells. However, GSH concentration in human plasma is relatively low (~ 0.01 mM), because of its rapid catabolism (Franco, et al., 2007). Consistent with the function as an antioxidant and detoxifier, GSH concentrations are particularly high in the liver (~ 10 mM), which is frequently exposed to xenobiotics, but also high concentrations can be found in the kidney, lungs and intestine. The GSH/GSSG ratio, which is often used as an indicator of the cellular redox state, is above 10 under normal physiological conditions in mammalian cells (Wu, et al., 2004). In microbial and plant cells, GSH is present with concentrations in the milimolar range as well. In E. coli cells, the ratio GSH/GSSG is very high and comes to 300-600 in cytoplasm. Interestingly, the extracellular concentration of GSH in the growth medium can be controlled by cells and modulated depending on bacterial growth conditions and phase, thus the extracellular GSH can probably play a role in physiology of bacteria (Smirnova & Oktyabrsky, 2005).
Since GSH is present in relatively high concentrations, sensitivity of quantitative analytical methods should not be critical for measurement of physiological GSH levels. However, high sensitivity is important when inhibitors of GSH synthesis are used or when determined in extracellular fluids. On the other hand, specificity (discrimination of GSH from other thiols present in cells) or selectivity (separation of GSH from possible interfering molecules in matrices) are required for an accurate GSH determination. In the case of GSSG, the method should always be very sensitive if measured in cells and extracellular compartments because of low concentrations of this compound (Camera & Picardo, 2002).
Many analytical tools are available for the analysis of GSH including non-separative spectrometric techniques, such as spectrophotometry, spectrofluorimetry, and a wide variety of separative methods (high performance liquid chromatography – HPLC, capillary electrophoresis – CE) allowing to measure GSH in different fluids such as cellular or tissue extracts, body fluids or microbial growth medium. Regardless of the method, in order to minimize oxidation and proteolysis of GSH during the sample handling, special precautions must be always taken (maintenance of pH below 7, refrigeration, protein precipitation by acidification) (Camera & Picardo, 2002).
Direct determination of GSH molecule with ultraviolet (UV) detection is possible, but highly devoid of sensitivity. Therefore, most of analytical approaches developed to assay GSH involves chemical derivatization of GSH followed by quantitation of the obtained adduct using an appropriate detection system. Incidentally, chromatographic and electrophoretic methods coupled with electrochemical detector or mass spectrometry (MS) enables very sensitive and specific GSH measurement without derivatization process. It is important to mention that common spectrometric, chromatographic and electrophoretic techniques typically measure the average concentration of GSH in a cell population after preparation of cell homogenates (GSH or tGSH level averaged across all cellular compartments). That is why, in situ techniques, such as fluorescence microscopy or flow cytometry (FC), have been developed in response to the need to analyze GSH in individual cells or in a specific cellular compartments.
As known, a derivatization reagent (label, dye, stain) introduces an appropriate tag (chromophore, fluorophore) into a molecule in order to permit its analysis with better detectability or separation properties. A good derivatization reagent should quickly and specifically react with the molecule and does not contribute to the matrix interference reactions and loss of sample during the reaction or does not require removing its excess prior to analysis. The obtained derivative must be relatively stable. In HPLC systems, this reaction can be applied as a pre-column or post-coulmn derivatization. However, derivatization process is an additional, often time-consuming step in sample preparation and the process can lead to varying and incomplete recovery of GSH (Rahman, et al., 2006). In the GSH molecule, the thiol moiety is susceptible to derivatization and preferred for its specificity compared to free carboxylic and amino functional groups present in this molecule (Camera & Picardo, 2002). Many derivatization reagents for GSH are available and their use has been already broadly described in the literature (Shimada & Mitamura, 1994). Nevertheless, a classical spectrophotometric assay with the use of 5’,5’-dithiobis(2-nitrobenzoic acid) (so-called Ellman’s reagent) and its modification (enzymatic recycling method) are until today widely used and have been accepted as routine for the determination of GSH in biological fluids like blood (Rahman, et al., 2006).
Most methods devoted to the measurement of GSH rely upon separative techniques like HPLC and CE (Shimada & Mitamura, 1994, Camera & Picardo, 2002). Chromatographic methods provide high degree of specificity and low detection levels, however, they imply time consuming handling, and are expensive and not readily available in most clinical laboratories. The enzymatic spectrophotometric assays are laborious and complicated. Spectrofluorimetric method is widely used in the field of biological science for its sensitivity and low cost, but, as a matter of fact, a required derivatization process needs an additional sample treatment. Moreover, all the techniques mentioned above are mostly characterized by a low sample throughput due to the sequential mode of analytical runs. However, a non-separative technique with a high sample throughput (fluorescence assay using the universal 96-well microplate format for the measurement of GSH in cells) have been also developed (Lewicki, et al., 2006). Nevertheless, new trends in the development of methods devoted to GSH monitoring are focused on its fluorescence labeling in living cells to allow GSH localization and/or determination using e.g. FC and confocal microscopy (in situ techniques). Therefore, there is a need for the development of GSH-selective fluorogenic or fluorescent probes.
In situ methods for glutathione and glutathione redox state imaging
Fluorescence imaging has been widely used in the life sciences for investigation of many cellular processes as it offers nanometer-scale resolution, fast, sensitive, reliable, and reproducible detection of ions, biomolecules and organelles in cells (Santra, et al., 2004, Resch-Genger, et al., 2008). However, the potential of a detection or imagining method is to a great extent determined by the properties of the probe used. Such a probe is a tag molecule designed to detect a particular component within a specific region of a biological specimen (including whole intact live cells, tissues) with adequate sensitivity and selectivity. To be suitable for these techniques, the probe must (a) be excitable without simultaneous excitation of the biological sample; (b) be detectable with conventional instrumentation; (c) posses a high molar absorption coefficient at the excitation wavelength; and (d) posses a high fluorescence quantum yield. Additionally, an ideal probe must exhibit non-toxicity, water solubility, stability, ability to penetrate intact cells, high reaction rate at neutral pH, and its adduct with a target molecule must have sufficient photostability (Resch-Genger, et al., 2008). A fluorescence probe is already a fluorophore, while a fluorogenic probe natively does not fluoresce, but forms a fluorescent adduct with a target molecule.
Owing to in situ methods, the target of a fluorescence probe can be localized in cells if examined under a fluorescence microscope. Whereas, application of fluorescence probes to cells followed by FC analysis allows subpopulations within a large sample to be identified and quantitated. Additionally, these in situ techniques even enable multicolor labeling experiments of cellular or subcellular components when simultaneously introduced two or more probes (Haugland, 2005). Some of reagents for GSH derivatization reacting with a thiol group are also useful as probes for GSH fluorescence imaging by means of in situ techniques. Fluorescent probes reported in the literature for in situ GSH imaging can be categorized into 3 major types: organic fluorogenic or fluorescent dyes, fluorophores of biological origin like genetically encoded fluorescent proteins and fluorescent nanoparticle probes (Wang, et al., 2006).
Organic fluorogenic or fluorescent probes for glutathione
A wide variety of thiol-reactive probes is available and used to label various cellular peptides, proteins and thiolated oligonucleotides for investigating their biological structure, function and interactions. The common thiol-reactive probes form with sulfhydryls chemically stable thioethers, e.g. maleimide dyes which are not appreciably fluorescent until after conjugation with thiols (Figure 4). On the other hand, there are thiol-reactive probes that have a structure of symmetric disulfide and undergo a thiol-disulfide interchange reaction to yield a new asymmetric disulfide. Reaction of most probes with thiols proceeds rapidly at room temperature in the physiological pH range (Haugland, 2005).
The general division of thiol-reactive probes into probes excited with visible light or UV light is used since such probes have very different chemical structures and reactive groups. In the class of the organic dyes excited with visible light, there are probes having visible absorption maxima beyond 410 nm, such as the Alexa Fluor, BODIPY, fluorescein and rhodamine derivatives. Then, the thiol-reactive probes exited with UV light have peak absorption below 410 nm, e.g. coumarin, naphthalene derivatives, dansyl aziridine and bimanes. Typically, UV light-excitable dyes exhibit blue fluorescence and less photostability than that of visible light-excitable dyes (Haugland, 2005).
Additionally, the fluorescent probes for GSH are categorized into GST-dependent and GST-independent probes. In both cases, adducts are formed between the dye and the sulfydryl group of GSH, resulting in a dramatic increase in fluorescence intensity and/or altered excitation/emission spectral properties of the dye. More specifically, GST-independent probes (non selective probes) react directly (non enzymatically) with the thiol group of GSH, e.g. monobromobimane (MBB), N-(1-pyrene)maleimide, BODIPY® maleimides (Haugland, 2005), and aromatic dialdehydic probes. The major weak point of these probes is a serious possibility of occurrence of side reactions with intracellular thiols other than GSH, such as low molecular weight compounds, e.g. cysteine, γ-GluCys and protein sulfydryls, often resulting in high levels of background fluorescence. However, GST-dependent probes for GSH (GSH selective or specific probes) contain a chloromethyl group and the formation of an adduct between the GSH molecule and a dye molecule is catalyzed by GST. Due to the fact that this binding is only possible in presence of GST, it has been conferred a high degree of specificity of these dyes to GSH with very low background labeling (Tauskela, et al., 2000). The following are examples of GST-dependent probes: monochlorobimane (MCB), 5-chloromethylfluorescein diacetate and 7-amino-4-chloromethylcoumarin.
Table of contents :
1. INTRODUCTION
2. THEORETICAL PART
2.1. Encapsulation of glutathione-selective fluorogenic probes
2.1.1. Biochemistry of glutathione
2.1.2. Functions of glutathione
2.1.3. Significance of glutathione in medicine and pharmacy
2.1.4. Analytical methods for glutathione detection and determination
2.1.5. In situ methods for glutathione and glutathione redox state imaging
2.1.5.1. Organic fluorogenic or fluorescent probes for glutathione
2.1.5.2. Redox-sensitive fluorescent proteins
2.1.5.3. Fluorescence nanoparticle probes
2.2. Encapsulation of salmon calcitonin
2.2.1. Physiologic action and clinical use of calcitonin
2.2.2. Structural properties and stability of calcitonin
2.2.3. Pharmaceutical preparations, side effects and pharmacokinetics
2.2.4. Novel calcitonin delivery systems and routes of administration
2.2.4.1. Parenteral sustained release calcitonin delivery systems
2.2.4.2. Oral calcitonin delivery systems
2.2.4.3. Other alternative routes of calcitonin administration
3. OBJECTIVES OF THE THESIS
4. EXPERIMENTAL PART
4.1. Encapsulation of glutathione-selective fluorogenic probes
4.1.1. Materials
4.1.2. Methods
4.1.2.1. Preparation of the probe-loaded nanoparticles
4.1.2.2. Characterization of the probe-loaded nanoparticles
4.1.2.3. In vitro release of the probe from nanoparticles
4.1.2.4. HPLC system and operating conditions
4.1.2.5. Pre-column derivatization of NDA
4.1.2.6. Study on NDA stability at various pH
4.1.2.7. Cell culture, probe loading and extraction conditions
4.1.2.8. Protein determination by the bicinchoninic acid method
4.1.2.9. Cytotoxicity studies
4.2. Encapsulation of salmon calcitonin
4.2.1. Materials
4.2.2. Methods
4.2.2.1. Preparation of salmon calcitonin-loaded nanoparticles
4.2.2.2. Characterization of salmon calcitonin-loaded nanoparticles
4.2.2.3. Study on salmon calcitonin stability
4.2.2.4. Study on salmon calcitonin adsorption to blank nanoparticles
4.2.2.5. In vitro salmon calcitonin release from nanoparticles
4.2.2.6. HPLC assay for salmon calcitonin analysis
4.2.2.7. DSC study
4.2.2.8. In vivo studies
4.2.2.9. Determination of salmon calcitonin in rat serum by ELISA
4.2.2.10. Spectrophotometric determination of calcium (II) in rat serum
4.2.2.11. Statistical analysis
5. RESULTS
5.1. Encapsulation of glutathione-selective fluorogenic probes
5.1.1. Validation of the HPLC method
5.1.2. Characterization of the probe loaded-nanoparticles
5.1.3. Loading of cells with NDA
5.1.4. Cytotoxicity of nanoparticles and NDA
5.2. Encapsulation of salmon calcitonin
5.2.1. Method validation of salmon calcitonin determination by HPLC
5.2.2. Method validation of salmon calcitonin determination by ELISA
5.2.3. In vitro characterization of calcitonin-loaded nanoparticles
5.2.4. Calcitonin in vitro stability and release from nanoparticles
5.2.5. In vivo studies
5.2.6. DSC study
6. DISCUSSION
6.1. Encapsulation of glutathione-selective fluorogenic probes
6.2. Encapsulation of salmon calcitonin
7. CONCLUSIONS
7.1. Encapsulation of glutathione-selective fluorogenic probes
7.2. Encapsulation of salmon calcitonin
8. REFERENCES
